Systems and methods for label-free detection of analytes

ABSTRACT

Disclosed are methods of detecting an analyte of interest comprising introducing a sample comprising an analyte of interest to an antibody or antibody fragment; incubating the sample and antibody or antibody fragment under conditions sufficient to allow binding of the analyte of interest to the antibody or antibody fragment; and detecting the binding of the analyte of interest to the antibody or antibody fragment using a label-free second harmonic detection system. Also disclosed are methods of screening and diagnosing using antibodies or antibody fragments and a label-free second harmonic detection system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 15/280,212, filed Sep. 29, 2016, which claims benefit of U.S.Provisional Application No. 62/284,412, filed Sep. 29, 2015, which ishereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01GM068120awarded by The National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

A simple cancer biomarker assay can provide early cancer detection andidentification so that individualized treatment strategies can bedelivered to patients in a timely manner. Such assays also have thepotential to track progression, regression, and recurrence of thedisease. When considering the multitude of potential cancer biomarkers,one area which has been largely overlooked is the low-molecular weight(LMW) distribution of peptides and protein fragments which are presentin blood, saliva and urine. These LMW species (haptens) are eitherdirectly secreted or are produced from the degradation of larger proteinproducts of cancer cells. LMW biomarkers have traditionally beenexcluded from development due to the difficulty in detecting thesespecies in an easy and reliable manner. The standard approaches used todate employ a competitive immunoassay with the hapten immobilized on asolid-support and binding of the primary antibodies to the surface is incompetition with the analyte (e.g. free hapten) in solution. Detectionis typically achieved using an enzyme-linked immunosorbant assay (ELISA)platform. Though this approach is the current “gold standard” inclinical assays, competitive assays suffer from a limited dynamic rangeand poor detection limits. In addition, the development of appropriatesecondary antibodies is also required and the method can also sufferfrom “antibody interference”. The detection and quantification of LMWcancer biomarkers in a label-free fashion using a primary antibodycapture assay would circumvent the limitations of current competitiveimmunoassays and would represent a significant advancement in theability to rapidly screen patients for potential cancers in a costeffective and efficient manner. BRIEF

SUMMARY

Disclosed are methods of detecting an analyte of interest comprisingintroducing a sample comprising an analyte of interest to an antibody orantibody fragment, incubating the sample and antibody or antibodyfragment under conditions sufficient to allow binding of the analyte ofinterest to the antibody or antibody fragment, and detecting the bindingof the analyte of interest to the antibody or antibody fragment using alabel-free second harmonic detection system.

Disclosed are methods of screening for analytes that bind an antibody orantibody fragment comprising introducing an analyte to an antibody orantibody fragment, and detecting the presence of the analyte bound tothe antibody or antibody fragment using a label-free second harmonicdetection system, wherein the presence of the analyte bound to theantibody or antibody fragment indicates the analyte binds the antibodyor antibody fragment.

Disclosed are methods of diagnosing cancer in a subject comprisingintroducing a sample obtained from a subject to an antibody or antibodyfragment, detecting the presence of an analyte bound to the antibody orantibody fragment using a label-free second harmonic detection system,diagnosing the subject with cancer when the presence of the analytebound to the antibody or antibody fragment is detected, andadministering a therapeutically effective amount of an anti-cancertreatment to the diagnosed subject.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIGS. 1A-1D show the detection of cocaine binding via the label-free SHimmunoassay and a control of methadone binding to an anti-cocaineantibody as a control. Second harmonic correlation spectroscopy (SHCS)analysis of the same binding experiment. Both methods obtain the samebinding data with the SHCS method also providing the association anddissociation rates in about 1/10th the time.

FIG. 2 is a comparison of the LOD for Second Harmonic (SH), SurfacePlasmon Resonance (SPR), Fluorescence and Enzyme Linked ImmunoSorbentAssay (ELISA).

FIG. 3A is a schematic of a SH heterodyning method disclosed herein.FIG. 3B depicts SH data for an untreated silica surface upon BSAadsorption and the SH heterodyned response from a surface with a KTPlayer for amplification in log scale.

FIG. 4 shows a list of candidate cancer biomarkers for targetedproteomics.

FIG. 5A shows a schematic illustration of an exemplary IgGimmobilization procedure, and FIG. 5B shows the creation of exemplaryantibody arrays using a Continuous Flow Microspotter (CFM) as disclosedherein.

FIGS. 6A-6B show the detection of Cocaine and Methadone in a primaryantibody assay. FIG. 6A shows the molecular structures of cocaine andmethadone (left) and the corresponding extinction coefficients (M-1cm-1) as a function wavelength (nm) (right), and FIG. 6B is a schematicof the immobilization procedure used to link the primary antibodies tothe solid support.

FIGS. 7A-7B show the detection of Cocaine and Methadone in a primaryantibody assay. FIGS. 7A shows the SH binding isotherms of cocaine andmethadone to anti-cocaine antibody immobilized on the sensor surface,and FIG. 7B is a schematic representation of the cocaine/anti-cocaineantibody capture event, with the corresponding Langmuir adsorptionisotherm model used to fit the data shown in FIG. 7A

FIG. 8 shows the SH binding isotherms of cocaine and methadone toanti-methadone antibody immobilized on the sensor surface

FIGS. 9A-9B show the second harmonic correlation spectroscopy (SHCS) ofCocaine and Methadone. FIG. 9A is the autocorrelation of cocaine bindingto anti-cocaine antibody measured using SHCS, and FIG. 9B is theautocorrelation of methadone binding to anti-methadone antibody measuredusing SHCS. The corresponding association (k_(on)) and dissociation(k_(off)) rates and the measured affinity constants (K_(a)) obtainedfrom the SHCS data are also displayed.

FIG. 10A depicts an exemplary second harmonic detection system includinga charge-coupled device detector. FIG. 10B depicts another exemplarysecond harmonic detection system including a photomultiplier tube and aprocessing assembly as disclosed herein. FIG. 10C schematically depictsa counter-propagating second harmonic generation configuration asfurther disclosed herein.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if an analyte of interest is disclosed anddiscussed, each and every combination and permutation of an analyte ofinterest and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

A. Definitions

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “asupport layer” includes a plurality of such support layers, reference to“the support layer” is a reference to one or more support layers andequivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “therapeutically effective amount” means anamount of a therapeutic, prophylactic, and/or diagnostic agent that issufficient, when administered to a subject suffering from or susceptibleto a disease, disorder, and/or condition, to treat, alleviate,ameliorate, relieve, alleviate symptoms of, prevent, delay onset of,inhibit progression of, reduce severity of, and/or reduce incidence ofthe disease, disorder, and/or condition, such as but not limited tocancer. In some instances, a therapeutically effective amount is anamount of a therapeutic that provides a therapeutic benefit to anindividual.

As used herein, the term “subject” or “patient” refers to any organismto which a composition of this invention may be administered, e.g., forexperimental, diagnostic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as non-human primates and humans;avians; domestic household or farm animals such as cats, dogs, sheep,goats, cattle, horses and pigs; laboratory animals such as mice, ratsand guinea pigs; rabbits; fish; reptiles; zoo and wild animals).Typically, “subjects” are animals, including mammals such as humans andprimates; and the like.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range¬from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

As used herein, the term “label-free” refers to a system or method thatis capable of providing information regarding molecular interactionswithout the need for an exogenous label.

As used herein, the term “analyte of interest” refers to a substancewhose chemical constituent is of interest in an analytical proceduresuch as a procedure to identify or measure the substance. An “analyte ofinterest” can be any substance, including, but not limited to, a nucleicacid, peptide, protein, antibody, or a small molecule.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.In particular, in methods stated as comprising one or more steps oroperations it is specifically contemplated that each step comprises whatis listed (unless that step includes a limiting term such as “consistingof”), meaning that each step is not intended to exclude, for example,other additives, components, integers or steps that are not listed inthe step.

B. Detection Systems

Disclosed are systems for detecting an analyte of interest using themethods described herein. Optionally, the system can be a secondharmonic detection system, such as, for example, a label-free secondharmonic detection system. Exemplary configurations of a label-freesecond harmonic detection system are disclosed herein. However, it iscontemplated that the disclosed methods can be performed using anyconfiguration of an optical imaging system that is capable of measuringand detecting second harmonic signals as disclosed herein.

In exemplary aspects, and with reference to FIGS. 12A-12C, thelabel-free second harmonic detection system can comprise a light source,such as a laser, that generates incident light at a first frequency.Optionally, the laser can be a Nd:YAG laser as is known in the art. Inexemplary aspects, it is contemplated that the laser can be a Nd:YAGpumped optical parametric oscillator (OPO) laser as is known in the art.In these aspects, it is further contemplated that the laser can betunable within a wavelength range of about 400 nm to about 2,000 nm. Insome aspects, it is contemplated that the incident light produced by thelaser can have a wavelength ranging from about 200 nm to about 1,000 nm.Thus, in exemplary aspects, the wavelength of the incident light canrange from about 200 nm to about 300 nm, from about 300 nm to about 400nm, from about 400 nm to about 500 nm, from about 500 nm to about 600nm, from about 600 nm to about 700 nm, from about 700 nm to about 800nm, from about 800 nm to about 900 nm, or from about 900 nm to about1,000 nm. Optionally, in exemplary aspects, the wavelength can be about532 nm. It is further contemplated that the optical parametricoscillator can be pumped with a selected pulse width ranging from about10 fs to about 10 ns and at a selected repetition rate ranging fromabout 1 Hz to about 1 MHz. It is contemplated that these parameters canbe selectively adjusted depending on system characteristics and processconditions. In use, it is contemplated that the laser can be pulsed in amanner such that the pulse width and pulse energy associated with theresulting beam provide a power density of least 100 kW/cm².

Optionally, as shown in FIGS. 12A-12B, the second harmonic detectionsystem can comprise a telescope assembly that receives a beam from thelaser (optionally, after the beam has passed through at least onepolarizer) and selectively adjusts (optionally, reduces) a diameter ofthe beam to a selected size (e.g., about 2 mm²).

In further aspects, the second harmonic detection system can furthercomprise a substrate assembly for supporting a sample. For example, insome aspects, the substrate assembly can have a prism layer and asupport layer that supports the sample between the support layer and theprism layer. In these aspects, it is contemplated that the portion ofthe prism layer in contact with the sample can be planar orsubstantially planar. In exemplary aspects, the prism layer can comprisean optical material that is transparent at both the excitation andsecond-harmonic wavelengths. Optionally, the prism layer can comprisesilicon oxide or fused silica; however, it is contemplated that othertypes of prisms can be used. In exemplary aspects, it is contemplatedthat the sample can be deposited on or otherwise secured to the prismlayer or the support layer using conventional methods. For example, inthese aspects, an aqueous solution containing the analtye of interestcan be placed in direct contact with the prism layer through the use ofa flow-cell or similar device as is known in the art. In use, thesubstrate assembly receives incident light at a first frequency from thelaser, and the incident light is reflected at the interface between thesupport layer and the sample. Optionally, the laser can be configured todirect light to the prism layer—and the prism layer can be oriented—suchthat the incident light is perpendicular or substantially perpendicularto an outer surface of the prism layer where the incident light entersthe prism. In exemplary aspects, the incident light can be delivered tothe prism layer at a selected angle of incidence ranging from about 5 toabout 85 degrees. Optionally, in various aspects and as shown in FIG.12C, it is contemplated that the support layer of the substrate assemblycan comprise a flow cell for performing spectroscopic measurements. Inthese aspects, the flow cell can be provided within a TEFLON block orother suitable chemically-resistant material to which the prism layer ismounted. Optionally, the flow cell can be provided with at least oneport (optionally, two ports on opposing sides of the block) to permitexchange of the cell volume. Optionally, in further aspects, the flowcell can be provided with additional ports for monitoring temperatureand pH as needed. In exemplary aspects, the flow cell can be providedwith at least one window that allow for detection of light at the secondharmonic wavelength through the window. Optionally, such a window can bepositioned on an opposed side of the flow cell from the sample. Thus, inaddition to detecting light at the second harmonic wavelength that isreflected from the top surface of the sample, it is contemplated thatthe disclosed system can be configured to detect light at the secondharmonic wavelength that is transmitted through the window. Optionally,the system can be configured to only detect light at the second harmonicwavelength that is transmitted through the window. Thus, it iscontemplated that any desired orientation of the substrate assembly canbe used.

Optionally, in additional aspects, the second harmonic detection systemcan comprise a mirror, such as a high-power dielectric mirror, thatreflects light back toward the sample (and the surface of the prismlayer supporting the sample) after incident light is reflected. As shownin FIG. 12C, the light reflected by the mirror can be provided at thesame wavelength as the incident light. In exemplary aspects, the angleof incidence of the light reflected by the mirror (toward the sample)can be equal to the angle of incidence of the light provided by thefirst light source.

Alternatively, in other optional aspects, the system does not include amirror. In these aspects, it is contemplated that the system cancomprise a second light source that delivers light at a frequency equalto the first frequency (delivered by the first light source). Inexemplary aspects, the angle of incidence of the light provided by thesecond light source can be equal to the angle of incidence of the lightprovided by the first light source.

The second harmonic detection system can further comprise a detectionassembly that receives reflected light from the substrate assembly afterthe incident light and the light reflected by the mirror (or the lightprovided by a second light source) contact the sample and cooperate togenerate a second harmonic signal. As disclosed herein, the detectionassembly can detect a second harmonic signal corresponding to reflectedlight having a second frequency equal to twice the first frequency. Inexemplary aspects, the detection assembly can comprise a monochromatoror other filtering element that is configured to remove scatteredfundamental light. In additional aspects, as shown in FIG. 12B, it iscontemplated that the detection assembly can comprise a photomultipliertube (PMT) that detects a second harmonic signal as further disclosedherein. Additionally or alternatively, it is contemplated that thedetection assembly can comprise a charge-coupled device (CCD) detectorthat is configured to detect a second harmonic signal as furtherdisclosed herein. In use, it is contemplated that the photomultipliertube and CCD can be configured to detect light at the second harmonicwavelength.

In further exemplary aspects, the second harmonic detection system cancomprise a processing assembly that receives the second harmonic signaland determines a quantity of an analyte of interest as further disclosedherein. In these aspects, the processing assembly can be communicativelycoupled (by wired or wireless connection) to the detection assembly.Optionally, in exemplary aspects, the processing assembly can comprise agated integrator and boxcar averager as are known in the art.Additionally, or alternatively, the processing assembly can comprise acomputing device, such as a computer, a tablet, a smartphone, a server,a network, a Cloud-based device, and the like, that is communicativelycoupled to the detection assembly. It is contemplated that the computingdevice can have processing circuitry that is coupled to a memory foraccessing software or data to assist with analysis of an analyte asdisclosed herein.

In further optional aspects, and as shown in FIG. 3A, the detectionassembly can comprise a sensor having a first surface that receivesreflected, second-harmonic light from the substrate assembly. It iscontemplated that the first surface of the sensor can comprise anysuitable material that is optically transparent at both the secondharmonic and excitation wavelengths. In these aspects, it iscontemplated that the first surface of the sensor can be opticallyheterodyned to amplify the second harmonic signal. For example, in oneexemplary non-limiting configuration, the first surface of the sensorcan be covered with a non-linear optically-active surface layer, such asfor example and without limitation, potassium titanyl phosphate (KTP).Optionally, the non-linear optically-active surface layer can bedirectly deposited onto the first surface of the sensor. The non-linearoptically-active surface layer can be covered with a silicon oxidesol-gel film to create a self-contained heterodyne optical arrangement.It is contemplated that the surface layers can be applied using aconventional sol-gel process as is known in the art. Alternatively, itis contemplated that the non-linear optically-active surface layer cancomprise a thin crystalline material or other non-linearoptically-active material that is attached to the first surface using anadhesive or bonding agent. In use, it is contemplated that theheterodyned sensor can be configured to enhance the second harmonicsignal as further disclosed herein.

C. Methods of Detecting

Disclosed are methods of detecting an analyte of interest comprisingintroducing a sample comprising an analyte of interest to an antibody orantibody fragment, incubating the sample and antibody or antibodyfragment under conditions sufficient to allow binding of the analyte ofinterest to the antibody or antibody fragment, and detecting the bindingof the analyte of interest to the antibody or antibody fragment using alabel-free second harmonic detection system.

In some instances, an analyte of interest can be a protein, nucleicacid, small molecule, or fragment thereof. In some instances, thefragment thereof of any of the protein, nucleic acid, or small moleculecan comprise the binding site for the disclosed antibody or antibodyfragment. In some instances, an analyte of interest can be a biomarker.For example, an analyte of interest can be a cancer biomarker. Forexample, a cancer biomarker can be, but is not limited to, BRCA1/BRCA2,CEA, EGFR, HER-2, PSA, S100, BCR-ABL, CA-125, p53, or p14ARF. In someinstances, an analyte of interest is a low molecular weight protein orfragment thereof. For example, low molecular weight proteins can be, butare not limited to, cytokines, chemokines, peptide hormones, orproteolytic fragments of larger proteins.

In some instances, a sample can be a biological sample. For example, abiological sample can be, but is not limited to, blood, serum, urine,milk, cell lysate, tissue lysate, plasma, tears, sweat, or ocular fluid.In some instances, a sample can be derived from a food source, whereinthe sample can be used for detecting contaminants.

In some instances, an antibody or antibody fragment is known to bind theanalyte of interest. In some instances, an antibody or antibody fragmentcan be immobilized on a support. For example, the support can be a solidsupport. In some instances, a solid support can be a solid-statesubstrate or support with which antibodies or antibody fragments can beimmobilized directly or indirectly. In some instances the antibodyfragment at least comprises the Fab portion of a full length antibody.In some instances the antibody fragment at least comprises the variableregion of a full length antibody.

Solid supports can include any solid material to which antibodies orantibody fragments can be coupled. This includes materials such asacrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene,polyethylene vinyl acetate, polypropylene, polymethacrylate,polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon,fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid,polylactic acid, polyorthoesters, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Solid supports can have anyuseful form including thin film, membrane, bottles, dishes, fibers,woven fibers, shaped polymers, particles, beads, microparticles, or acombination. Solid supports can be porous or non-porous. A form for asolid-state substrate is a microtiter dish, such as a standard 96-welltype.

Methods for immobilizing antibodies (and other proteins) to solidsupports are well established. Immobilization can be accomplished byattachment, for example, to aminated surfaces, carboxylated surfaces orhydroxylated surfaces using standard immobilization chemistries.Examples of attachment agents are cyanogen bromide, succinimide,aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents,epoxides and maleimides. A preferred attachment agent is theheterobifunctional cross-linker N-[γ-Maleimidobutyryloxy] succinimideester (GMBS). These and other attachment agents, as well as methods fortheir use in attachment, are described in Protein immobilization:fundamentals and applications, Richard F. Taylor, ed. (M. Dekker, NewYork, 1991);, Johnstone and Thorpe, Immunochemistry In Practice(Blackwell Scientific Publications, Oxford, England, 1987) pages 209-216and 241-242, and Immobilized Affinity Ligands; Craig T. Hermanson etal., eds. (Academic Press, New York, 1992) which are incorporated byreference in their entirety for methods of attaching antibodies to asolid support. Antibodies can be attached to a support by chemicallycross-linking a free amino group on the antibody to reactive side groupspresent within the solid support. For example, antibodies may bechemically cross-linked to a support that contains free amino, carboxyl,or sulfur groups using glutaraldehyde, carbodiimides, or GMBS,respectively, as cross-linker agents. In this method, aqueous solutionscontaining free antibodies are incubated with the solid support in thepresence of glutaraldehyde or carbodiimide.

A method for attaching antibodies or other proteins to a solid supportis to functionalize the support with an amino- or thiol-silane, and thento activate the functionalized support with a homobifunctionalcross-linker agent such as (Bis-sulfo-succinimidyl suberate (BS3) or aheterobifunctional cross-linker agent such as GMBS. For cross-linkingwith GMBS, glass supports are chemically functionalized by immersing ina solution of mercaptopropyltrimethoxysilane (1% vol/vol in 95% ethanolpH 5.5) for 1 hour, rinsing in 95% ethanol and heating at 120° C. for 4hrs. Thiol-derivatized slides are activated by immersing in a 0.5 mg/mlsolution of GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at roomtemperature. Antibodies or proteins are added directly to the activatedsupport, which are then blocked with solutions containing agents such as2% bovine serum albumin, and air-dried. Other standard immobilizationchemistries are known by those of skill in the art.

Disclosed are methods of detecting an analyte of interest comprisingintroducing a sample comprising an analyte of interest to an antibody orantibody fragment, incubating the sample and antibody or antibodyfragment under conditions sufficient to allow binding of the analyte ofinterest to the antibody or antibody fragment, and detecting the bindingof the analyte of interest to the antibody or antibody fragment using alabel-free second harmonic detection system, wherein the concentrationof the analyte in the sample is sub-femtomolar. In some instances, theconcentration of the analyte in the sample can be sub-micromolar,sub-nanomolar, sub-picomolar, or sub-femtomolar. In some instances, theconcentration of the analyte of interest in the sample can be micromolarto sub-femtomolar.

Disclosed are methods of detecting an analyte of interest comprisingintroducing a sample comprising an analyte of interest to an antibody orantibody fragment, incubating the sample and antibody or antibodyfragment under conditions sufficient to allow binding of the analyte ofinterest to the antibody or antibody fragment, and detecting the bindingof the analyte of interest to the antibody or antibody fragment using alabel-free second harmonic detection system, further comprising a stepof washing away any unbound analyte prior to detecting the binding ofthe analyte of interest.

In some aspects, the disclosed composition and methods can be used indrug screening of a subject. For example, antibodies or antibodyfragments capable of binding to a drug or drug metabolite can be used inthe methods disclosed herein. As shown herein, methods of detectingcocaine and methadone in a sample can be performed using the methodsdisclosed herein.

In some aspects, the disclosed composition and methods can be used todetect a contaminant in a food. For example, a sample from a food sourcecan be screened using the methods disclosed herein to determine thepresence of a particular analyte of interest (e.g. a contaminant).

1. Introducing a sample comprising an analyte of interest to an antibodyor antibody fragment;

In some instances, introducing a sample comprising an analyte ofinterest to an antibody or antibody fragment comprises adding the samplecomprising an analyte of interest to the antibody or antibody fragment.In some instances, the antibody or antibody fragment can be in asolution, immobilized to a support or a combination thereof.

In some instances, introducing a sample comprising an analyte ofinterest to an antibody or antibody fragment comprises adding theantibody or antibody fragment to a sample comprising an analyte ofinterest. In some instances, the antibody or antibody fragment bound tothe analyte of interest can be subsequently immobilized to a supportprior to detection.

2. Incubating the sample and antibody or antibody fragment underconditions sufficient to allow binding of the analyte of interest to theantibody or antibody fragment; and

In some instances, incubating the sample and antibody or antibodyfragment under conditions sufficient to allow binding of the analyte ofinterest to the antibody or antibody fragment comprises providingincubation conditions such as appropriate temperature, pH and saltconcentration to allow binding of the analyte of interest to theantibody or antibody fragment. In some instances, incubating the sampleand antibody or antibody fragment under conditions sufficient to allowbinding of the analyte of interest to the antibody or antibody fragmentcomprises providing appropriate concentrations of the antibody orantibody fragment to bind the analyte of interest.

3. Detecting the binding of the analyte of interest to the antibody orantibody fragment using a label-free second harmonic detection system.

In some instances, detecting the binding of the analyte of interest tothe antibody or antibody fragment using a label-free second harmonicdetection system makes use of non-linear optical methods such assecond-harmonic imaging (SHI) and second-harmonic correlationspectroscopy (SHCS) to produce a detection scheme for a universal,label-free immunoassay as further disclosed herein.

In exemplary aspects, detecting the binding of the analyte of interestto the antibody or antibody fragment using the label-free secondharmonic detection system can comprise using second harmonic imaging(SHI) to detect the binding of the analyte of interest. In additionalaspects, detecting the binding of the analyte of interest to theantibody or antibody fragment using the label-free second harmonicdetection system can further comprise determining binding properties ofthe analyte of interest using second-harmonic correlation spectroscopy.Optionally, detecting the binding of the analyte of interest to theantibody or antibody fragment using the label-free second harmonicdetection system can comprise using second-harmonic correlationspectroscopy to determine binding affinity data (e.g., K_(a), K_(d), andthe like) for the analyte of interest based upon a single measuredconcentration of the analyte of interest. The second-harmonic signal canbe recorded as a function of time at a single analyte concentration. Theresulting signal can then be correlated automatically to retrieve thedynamics of the binding process. The resulting correlation is thennumerically fit to the follow equation:

${G(\tau)} = {\frac{k_{off}}{k_{on}\lbrack A\rbrack} \times {\exp\lbrack {{- ( {{k_{on}\lbrack A\rbrack} + k_{off}} )}\tau} \rbrack}}$

Using sing a nonlinear least squares regression algorithm, theparameters, k_(on) and k_(off) can be obtained. Additionally, detectingthe binding of the analyte of interest to the antibody or antibodyfragment using the label-free second harmonic detection system canfurther comprise using second-harmonic imaging to quantify the analyteof interest. The second-harmonic imaging can be accomplished by directcollection of a wide field image using a CCD device using a largeillumination area which covers the entirety or a fraction of the sensorarea. Alternatively, the second-harmonic imaging can be accomplished byraster scanning of the incident excitation source over the surface insuch a way as to construct and image from individual collection pointsbased on the location of the excitation source on the surface using aPMT or other suitable photon detector system.

Optionally, in additional aspects and as previously described, thelabel-free second harmonic detection system can comprise: a laser thatgenerates incident light at a first frequency; a substrate assemblyhaving a prism layer and a support layer that supports the sample andantibody or antibody fragment between the support layer and the prismlayer, wherein the substrate receives incident light at the firstfrequency from the laser; a detection assembly that receives reflectedlight from the substrate assembly, wherein the detection assemblydetects a second harmonic signal corresponding to reflected light havinga second frequency equal to twice the first frequency; and a processingassembly that receives the second harmonic signal and determines aquantity of the analyte of interest. In further optional aspects, and aspreviously described, it is contemplated that the detection assembly cancomprise a sensor having a first surface that receives reflected lightfrom the substrate assembly. In these aspects, it is contemplated thatthe first surface of the sensor can be optically heterodyned to amplifythe second harmonic signal. For example, in one exemplary non-limitingconfiguration, the first surface of the sensor can be covered with anon-linear optically-active surface layer, and the non-linearoptically-active surface layer can be covered with a silicon oxidesol-gel film to create a self-contained heterodyne optical arrangementthat enhances the second harmonic signal.

D. Methods of Screening

Disclosed are methods of screening for analytes that bind an antibody orantibody fragment comprising introducing an analyte to an antibody orantibody fragment, and detecting the presence of the analyte bound tothe antibody or antibody fragment using a label-free second harmonicdetection system, wherein the presence of the analyte bound to theantibody or antibody fragment indicates the analyte binds the antibodyor antibody fragment.

In some instances, an antibody or antibody fragment is known to bind theanalyte of interest. In some instances, an antibody or antibody fragmentcan be immobilized on a support. For example, the support can be a solidsupport. In some instances, a solid support can be a solid-statesubstrate or support with which antibodies or antibody fragments can beimmobilized directly or indirectly. In some instances the antibodyfragment at least comprises the Fab portion of a full length antibody.In some instances the antibody fragment at least comprises the variableregion of a full length antibody.

In some instances, an analyte of interest can be a protein, nucleicacid, small molecule, or fragment thereof. In some instances, thefragment thereof of any of the protein, nucleic acid, or small moleculecan comprise the binding site for the disclosed antibody or antibodyfragment. In some instances, an analyte of interest can be a biomarker.For example, an analyte of interest can be a cancer biomarker. Forexample, a cancer biomarker can be, but is not limited to, BRCA1/BRCA2,CEA, EGFR, HER-2, PSA, S100, BCR-ABL, CA-125, p53, or p14ARF. In someinstances, an analyte of interest is a low molecular weight protein orfragment thereof. For example, low molecular weight proteins can be, butare not limited to, cytokines, chemokines, peptide hormones, orproteolytic fragments of larger proteins.

In exemplary aspects, detecting the presence of the analyte bound to theantibody or antibody fragment using the label-free second harmonicdetection system can comprise using second harmonic imaging (SHI) todetect the binding of the analyte to the antibody or antibody fragmentas further disclosed herein. In further exemplary aspects, it iscontemplated that detecting the presence of the analyte bound to theantibody or antibody fragment using the label-free second harmonicdetection system can comprise determining binding properties of theanalyte of interest using second-harmonic correlation spectroscopy asfurther disclosed herein.

E. Methods of Diagnosing

Disclosed are methods of diagnosing cancer in a subject comprisingintroducing a sample obtained from a subject to an antibody or antibodyfragment, detecting the presence of an analyte bound to the antibody orantibody fragment using a label-free second harmonic detection system,diagnosing the subject with cancer when the presence of the analytebound to the antibody or antibody fragment is detected, andadministering a therapeutically effective amount of an anti-cancertreatment to the diagnosed subject. In some instances, the subject ishuman.

In some instances, a sample can be a biological sample. For example, abiological sample can be, but is not limited to, blood, serum, urine,milk, cell lysate, tissue lysate, plasma, tears, sweat, or ocular fluid.

In some instances, an analyte of interest can be a protein, nucleicacid, small molecule, or fragment thereof. In some instances, thefragment thereof of any of the protein, nucleic acid, or small moleculecan comprise the binding site for the disclosed antibody or antibodyfragment. In some instances, an analyte of interest can be a biomarker.For example, an analyte of interest can be a cancer biomarker. Forexample, a cancer biomarker can be, but is not limited to, BRCA1/BRCA2,CEA, EGFR, HER-2, PSA, S100, BCR-ABL, CA-125, p53, or p14ARF. In someinstances, an analyte of interest is a low molecular weight protein orfragment thereof. For example, low molecular weight proteins can be, butare not limited to, cytokines, chemokines, peptide hormones, orproteolytic fragments of larger proteins.

In some instances, the anti-cancer treatment can be any knownanti-cancer treatment including, but not limited to, chemotherapy,radiation, immunotherapy, cell therapy, or hormone therapy.

In exemplary aspects, detecting the presence of the analyte bound to theantibody or antibody fragment using the label-free second harmonicdetection system can comprise using second harmonic imaging (SHI) todetect the binding of the analyte to the antibody or antibody fragment.In further exemplary aspects, detecting the presence of the analytebound to the antibody or antibody fragment using the label-free secondharmonic detection system can comprise determining binding properties ofthe analyte of interest using second-harmonic correlation spectroscopy.

F. Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for detecting ananalyte of interest, the kit comprising an antibody or antibody fragmentand at least one component of a label-free second harmonic detectionsystem. The kits also can contain a solid support.

The disclosed kits can also include instructions for how to use thelabel-free second harmonic detection system.

EXAMPLES

Cancer biomarkers have the potential of providing a reliable detectionscheme for early cancer detection, identification and progression,allowing patients to receive the most effective and appropriatetherapies. The presence of cancer in a patient generates a uniquesignature in the blood, through the presence of LMW proteins andpeptides originating from cancer cells in the body. A multitude ofspecific biomarkers and their effective concentrations in the blood needto be evaluated to provide an accurate assessment of the state, type andprogression of cancer in a patient. The goal of this proposal is not toidentify such markers, but to implement a feasible multiplexed arraybased detection assay for quantification and identification of suchbiomarkers in a clinical setting.

There have been a number of analytical approaches taken to detect andquantify cancer biomarkers in blood and other biological samples,including the polymerase chain reaction (PCR), capillaryelectrophoresis, surface plasmon resonance (SPR), surface enhanced Ramanspectroscopy (SERS), microcantilevers, colorimetric assays,electrochemical assays, and a variety of fluorescence methods. Theseapproaches and others have been summarized in a number of recentreviews. Competitive enzyme-linked immunosorbent assays (ELISAs), havealso been employed, which is not surprising due to their extensivelyused in clinical diagnostics for decades. They are also considered thegold standard for the detection of proteins in physiological samples bymany.

Traditionally, detection of LMW substances in blood, salvia or urinehave been carried out using competitive heterogeneous ELISA basedimmunoassays employing immobilized antibodies or small-moleculeconjugates on a solid support. Competitive immunoassays are simple,rapid and cost-effective methods for detecting a wide range of LMWanalytes in variety of biological matrixes. However, the number ofcommercially available immunoassay kits for haptens is relativelylimited. The surprising absence of antibody based assays for haptens inthe marketplace is due largely to the poor performance (reproducibility)and high limits of detection (LOD) typically associated with the currenttests. An alternative would be to use a noncompetitive assay. A studyreviewing the fundamental problems associated with competitiveimmunoassays showed that they are inferior to noncompetitiveimmunoassays in terms of sensitivity, precision, kinetics and dynamicrange of the analyte. However, a practical noncompetitive haptenimmunoassay format has not been forthcoming, due to the difficulty ofdetecting the hapten upon capture. Clearly, the development of anoncompetitive cancer biomarker assay would overcome the limitations ofexisting competitive ELISA tests, but the detection problem is thelargest hurdle to overcome in developing a functional noncompetitive orprimary antibody assay.

1. Developing a Label-Free Noncompetitive Cancer Biomarker Immunoassay

The prevalence of cancer and the need for early detection makes thedevelopment of accurate, rapid, cost effective assays of paramountimportance. The goal here is to apply SHCS and SHI to develop alabel-free noncompetitive immunoassay for cancer biomarkers. Initialstudies allow for method validation of the approach and characterizationin biological matrices (bovine plasma or urine) can prove the viabilityof the assay in a clinically relevant platform. These studies present acomprehensive examination of the efficacy of SHCS and Second HarmonicImaging (SHI) for the label-free detection of cancer biomarkers in aprimary antibody immunoassay format. These studies can advance theability to detect a host of biologically relevant molecules andbiomarkers associated with cancer, significantly improving upon existingclinical assays.

It is estimated that greater than 1 million people annually arediagnosed with cancer of some form. The high prevalence of cancer andthe need for early detection makes the development of accurate, rapid,cost effective and widely available assays of paramount importance. Theability to detect low concentrations of cancer biomarkers (fM and lower)is also highly desirable. From an analytical perspective, lower LODslead to earlier detection and more favorable outcomes for patients.

A recent examination of bottlenecks in the development of clinicalassays for cancer detection based on biomarkers found that the reasonbehind so few biomarkers reaching the clinic can largely be explained bythe inability of current technologies to consistently and quantitativelyverify the presence of the candidates (biomarkers) in patient samples.Clearly an accurate, highly sensitive, label-free noncompetitiveimmunoassay would be of clinical relevance for the screening ofpotential cancer biomarkers.

There are approximately 1200 proteins which have been identified aspotential biomarkers for various forms of cancer. However, only 9 havebeen FDA approved for cancer screening. In order to validate SHCS andSHI for biomarker detection, 6 FDA approved biomarkers (FIG. 4 ) will bescreened in a primary antibody array using SHI and SHCS. Thesecandidates were chosen principally because existing working assays existwith established LODs. The proteins and primary antibodies needed tocreate the arrays are also commercially available.

The approach described herein uses the nonlinear optical process ofsecond harmonic (SH) generation for the label-free detection of cancerbiomarkers in an immunoassay format. SH is a coherent second-ordernonlinear optical technique which is inherently surface-specific andpossesses the spectroscopic characteristics of UV-Vis absorbancespectroscopy. The principles of SH have been discussed in a number ofexcellent reviews. SH involves the frequency doubling of an incidentoptical field at frequency w yielding photons at twice the frequency(2ω) of the incident source (FIG. 10C). The frequency doubling can bemodeled using the following equation: 2ω=ω_(vis)+ω_(vis). The measuredSH intensity is proportional to the square of the surface density ofmolecules; coupled with the surface-specificity of the technique, makesit ideally suited for detection in a heterogeneous assay. The surfaceselectivity eliminates the need for a “washing” step, allowing a trueequilibrium measurement to be made, which is crucial when sub-micromolar(μM) concentrations of analytes are measured and overcomes the largestsource of error in current competitive ELISA based immunoassays. If thefundamental (ω) or SH (2ω) frequencies are resonant with electronictransitions of the small-molecule, peptide or protein of interest, anincrease in the SH signal is observed. The SH signal can be modeledaccording to the following equation:

$\begin{matrix}{{\chi_{R}^{(2)} = {\sum\limits_{i}\frac{NA_{i}}{\omega_{i} - \omega_{laser} - {i\Gamma_{i}}}}},} & \end{matrix}$

where ω_(j)=resonant transition in UV or Vis.

As almost all small-molecules of biological importance (peptides,proteins or metabolites) contain double bonds or conjugated ring systemswith electronic transitions in the UV and deep UV, a considerable SHenhancement is achieved when using a visible pump. The versatility of SHfor the label-free detection of various small-molecule drugs, peptidesand proteins to model lipid membranes was previously established. SHtechniques are also exquisitely sensitive to molecular symmetry; assuch, a random orientation of molecules produces no measurable SH due tothe symmetry constraints of the up-conversion process. However, when amolecule is specifically bound to a receptor, such as an antibody, thenet alignment of the molecules allows for detection (FIG. 1 ), thuseliminating the need for sophisticated blocking buffers as required inmost ELISA-based assays. The capacities of SH have also been extended bydeveloping a new imaging modality. Coupling SHI with a microarrayfabrication method using a continuous flow micro-spotter allows for thedevelopment of a high-throughput label-free cancer biomarker immunoassayas disclosed herein.

i. Continuous Flow Microspotter for Array Formation

The use of arrays for chemical/biological analysis is well proven, andprovides a high-throughput or multiplex advantage. A Continuous FlowMicrospotter (CFM) is ideally suited for the creation of biological andmolecular arrays. Two unique applications of the CFM are 1) the creationof multicomponent lipid bilayer arrays (MLBAs) and 2) the demonstrationof protein capture. The CFM can be used to create discrete lipidbilayers containing a variety of ligands for the selective binding ofprotein receptors. MLBAs containing lipids functionalized with the GM1ganglioside, DNP and biotin were screened with fluorescently labeledcholera toxin B (CTB), anti-dinitrophenyl (DNP) antibody andNeutrAvidin, in a protein-capture array. The arrays show little to nocross-reactivity and excellent spot-to-spot reproducibility.

ii. Protein and Small-Molecule Detection Using SH

Most conventional microarray-based strategies for detecting proteins,peptides and some small-molecules are based on fluorescence. SH and SHIhave been developed to quantify such interaction in a label-free manner.The predominant chromophore in proteins and peptides is the amidebackbone. Ignoring contributions from aromatic side chains, this groupconstitutes the major source of the SH response due to the relativelylarge nonlinear polarizabilitiy of the 7C electrons. Below are severalexamples of successes in implementing SH for the detection of proteinsand peptides, the detection of small-molecule drugs, and the developmentof SHI coupled with CFM created arrays for the high-throughput screeningof drug-membrane interactions.

a. SH Detection of Proteins and Peptides

Biotin-bound protein complexes have been used in a wide variety ofbioanalytical applications, including monitoring conformational changes,biochip sensor fabrication, immunoassays, and targeted drug delivery andscreening. A comparison of the binding properties of avidin,streptavidin, neutrAvidin™ and anti-biotin antibody to a biotinylatedlipid bilayer was studied using SH, providing a direct comparison of thebinding properties of these biotin-protein complexes in a label-freemanner and providing an unbiased comparison of the binding affinities.

b. Quantifying Drug-Membrane Interactions

SH was also used to measure the association of several selectiveestrogen receptor modulator (SERM) drugs to lipid bilayers, tounderstand how the interaction between the SERMs cell membrane modulatesthe drugs bioavailability. Tamoxifen and raloxifene are the most widelyprescribed SERMs to treat breast cancer and prevent osteoporosis. Thebinding of raloxifene, tamoxifen and three tamoxifen metabolites toseveral artificial cell membranes were detected and the differences inmembrane interactions for these SERMs and their metabolites werequantified for the first time, and these findings were correlated withtheir clinical potency.

c. SH Imaging of Small-Molecule Adsorption to Membranes

SH is a powerful tool for investigating small-molecule and proteininteractions with membranes in a label-free manner. However, ahigh-throughput analog would be even more advantageous, akin tofluorescence imaging. Towards that end, SHI was implemented to measurethe interactions between the local anesthetic tetracaine and amulti-component lipid bilayer array (MLBA) in a label-free mannerallowing the effects of lipid phase and CHO content on tetracainebinding to be examined simultaneously. SHI shows that tetracaine has ahigher binding affinity to l.c. phase lipids than to solid-gel phaselipids. The maximum surface excess of tetracaine increases with thedegree of unsaturation of the phospholipids and decreases withcholesterol in the lipid bilayers. This study demonstrates that SHIpossesses the required sensitivity to directly quantify biomarkers in ahigh-throughput manner.

iii. Second-Harmonic Correlation Spectroscopy

Determining binding kinetics and subsequent binding affinities inbioassays is technically challenging and time intensive. Typicallyadsorption and desorption rates are measured in real time, such as inSPR. Another approach which has been used in fluorescence spectroscopyis to measure the fluctuations in a system for a fixed time and throughcorrelation analysis, retrieve the underlying kinetic rates. Althoughfluorescence correlation spectroscopy (FCS) has been extensivelydeveloped and implemented for such surface interactions, theinterpretation of the fluorescence autocorrelation data is complicatedby fluorescence arising from species in solution, backgroundfluorescence, and photobleaching.

We have applied the principles of FCS to the label-free and surfacespecific technique of SH for the first time, overcoming many of thelimitations of FCS. Second harmonic correlation spectroscopy (SHCS)eliminates observable fluctuations from molecular diffusion in solutionand ameliorates problems associated with the degradation of fluorophoreswithout having to reduce the number of molecules in the observationarea, resulting in faster acquisition times and simplified dataanalysis.

To demonstrate the capabilities of SHCS, the binding kinetics of(S)-(+)-1,1′-bi-2-napthol (SBN) associating with a DOPC lipid bilayerwere examined. The results obtained from SHCS were compared to aclassical adsorption isotherm experiment to verify the accuracy of thekinetic and thermodynamic values retrieved from the SHCS analysis. UsingSHCS, both the adsorption and desorption rates were simultaneouslydetermined in a much shorter time compared to a conventional isothermstudy. Additionally, SHCS was used to deconvolute the complex bindingproperties of multivalent protein-ligand interactions (peanut agglutinin(PnA) and cholera toxin subunit b (CTb) binding to a GM1 which wasinaccessible using conventional binding isotherms, SHCS provides a moreefficient and comprehensive method for studying molecular interactionsat a surface without the need for an exogenous label, providing a newand powerful method for investigating cancer biomarker detection usingantibodies, as discussed below.

iv. A Proven Small-Molecule Noncompetitive Label-Free Immunoassay

The use of antibodies for the direct detection of LMW small-moleculeswas not possible using a primary antibody immunoassay format; that isuntil now. SH and SHCS can detect cocaine (303.4 MW) in a noncompetitivelabel-free immunoassay with high sensitivity and selectivity (FIG. 1 ).The LOD is dependent upon the sensitivity (proportional to K_(a)) andnoise (σ). Based on the data in FIG. 1 , SH has a 20× lower LOD(0.13±0.07 nM) compared to a standard competitive ELISA (2.9±1 nM). SHCSalso provides a rapid assessment of the kinetics ofadsorption/desorption and thermodynamic affinity comparable to theisotherm data in a fraction of the time (FIG. 1 ). The example shown inFIG. 1 for small-molecule capture in a noncompetitive label-free assayusing SH illustrates the potential this method has for cancer biomarkerdetection. Impressive as SH is for the label-free detection of LWMcompounds in a noncompetitive immunoassay, improvement of thesensitivity and reducing error in the measurement are crucial for acancer biomarker assay in order to improve the LOD further. The goal ofthis research is to reduce the LOD to the fM range for cancer biomarkersdetection. The proposed research describes a strategy to achieve thisobjective by implementing a SH enhancement scheme as disclosed herein.

v. Formation of Antibody Arrays Using the CFM

Following an established protocol published by Pierce Biotechnology(Tech Tip #5, Thermo Scientific,piercenet.com/files/TR0005-Attach-Ab-glass.pdf, FIG. 5 ), IgG proteinsagainst the various compounds listed in FIG. 4 , will be arrayed ontofused silica supports. A silica surface functionalized with3-aminopropyltrimethoxy silane is reacted with a heterobifunctionalcross linker (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid3-sulfo-N-hydroxysuccinimide ester sodium salt, i.e. Sulfo-SMCC). Thesulfhydryl groups (—SH) on the antibody are made available for covalentcoupling to the maleimide-activated surface by reacting native disulfidebonds of the antibody with 2-mercaptoethylamine. Thesulfhydryl-containing antibodies can be cross-linked to themaleimide-activated silica surface using the CFM.

vi. Screening Cancer Biomarkers

The representative cancer biomarkers can be reconstituted in PBS, at pH7.4 and incubated with a primary antibody array for detection. SHCS canbe used to calibrate the capture antibody arrays by determining thebinding association constant for each of the primary antibody-targetbiomarker pairs. Using this calibration data, the LOD for the variousbiomarkers can be determined. The measurement step can be performedusing SHI. Cross-reactivity can be examined using antibody arrays forthe various markers screened in unison. These measurements can be usedto ascertain the specificity of the various capture antibodies for thespecific cancer biomarker target.

vii. Screening Libraries in Biologically Relevant Media

As with the small-molecule immunoassays described in above, thecalibration and measurement of biomarker association to the primaryantibody arrays can be reproduced in bovine plasma and synthetic urine.

viii. Validation of SHCS and SHI

The effectiveness of these methods for the screening of the selectedcancer biomarkers can be accomplished in two ways: 1) The SHC and SHIapproach can be compared to the results obtained using commerciallyavailable assays. The LODs, sensitivity and selectively can be measuredand compared with the established assays. 2) The ESHC and ESHI resultscan also be correlated with fluorescence measurements made on the samesystems. For the protein biomarker studies, fluorescein (Fl)- orrhodamine (Rh)-labeled analogs of the proteins can be used. Labeling canbe accomplished with amine reactive conjugates (lissamine rhodamine Band fluorescein succinimidyl esters, available from Invitrogen).Fluorescence microscopy can be used to collect the binding data. Thekinetic and steady-state adsorption data obtained by fluorescence can becorrelated with that obtained from the SH experiments as a means ofvalidating the SHCS and SHI methods. A statistical assessment of thebinding affinities between formats can also be performed as a means ofvalidating the SH detection schemes.

2. Enhancing the Capabilities of Second Harmonic for Cancer BiomarkerDetection

One goal of the proposed studies is to enhance and implement thecomplimentary techniques of SHCS and SHI for the development of auniversal, label-free immunoassay for a range of potential LMW cancerbiomarkers. SHCS can be implemented for rapid single point calibrationto derive the Kd (or Ka) in a fraction of the time required by aconventional thermodynamic equilibrium binding isotherm. This can becoupled with the high-throughput capabilities of SHI for screening andquantifying small-molecules by utilizing antibody capture arrays.Together these methods represent a unique analytical approach which doesnot compromise sensitivity and speed yet provides a novel label-freeimmunoassay format. Alone, the techniques are already proven effectivelabel-free methods for detecting proteins and small-molecules, but inorder to tackle the changes of detecting cancer biomarkers at thelow-levels found in biological samples, the sensitivity of these methodsneeds to be improved. To achieve this, enhancing these already capabletechniques using a novel optical heterodyning method, providing greatersensitivity and improved limit of detection (LOD) can be performed.

The detection limit achievable with the current SH methods is comparedwith competing technologies SPR, fluorescence, and ELISA (FIG. 2 ). Thedata highlights the impressive sensitivity of SH and the clear advantageit has in the label-free detection of both small-molecules and proteins.The studies from which the data in FIG. 2 were obtained are summarizedabove. As seen in FIG. 2 , SH surpasses the detection limits of currentstate-of-the-art analytical approaches. In most cases, SH has a LOD thatis several orders of magnitude better than existing techniques. Althoughimpressive, the LODs still needs to be improved in order to provide aplatform capable of detecting the sub-femtomolar (fM) concentrations orlower of cancer biomarkers.

The limited conversion efficiency of nonlinear optical phenomena such asSH underscores the importance of signal enhancement strategies.Enhancement strategies also have the potential to move SH from thelaboratory to the clinic by utilizing compact low-cost (˜$5K), lasersources. One method which has been used to increase the “signal” in hasbeen optical heterodyning where an external field is “added” to theintrinsically weak SH signal providing significant amplification. Theintensity at 2ω (I_(2ω)) for a heterodyned system is given by thefollowing: [I_(2ω)≈(E_(LO))²+2E_(LO)E_(SH)exp(−iϕ)+E_(SH) ²] whereE_(LO) is the electric field produced by the local oscillator (LO),E_(SH) is the electric field of the SH generated from the moleculesadsorbed at the surface and ϕ is the phase difference between LO and SH.E_(SH)=N<β_(ijk)>E(ω)², with N being the surface density of analyte,β_(ijk) is the hyperpolarizability, and E(ω) is the electric fieldamplitude of the fundamental field (ω). As E_(LO) is large, the lastterm in the equation can be negated. The intensity at 2ω is dominated bythe LO and the product of the LO and SH fields. As the LO is constant itcan be subtracted from the measured intensity, giving an amplified SHresponse linearly dependent upon the concentration of analyte.

Typically optical heterodyning is accomplished with a LO which isgenerated external to the system. This approach is cumbersome andrequires precise optical alignment of the LO with the SH generated atthe surface in order to maintain the optimal phase relationship betweenthe LO and SH fields. One exemplary approach, as further disclosedherein, has been to deposit a nonlinear optically-active surface layer,such as potassium titanyl phosphate (KTP), directly onto the sensorsurface using the sol-gel process. The 10 nm thick KTP layer is thenover-coated with a SiO₂ sol-gel film creating a self-containedheterodyne optical arrangement (shown schematically in FIG. 3 ). Theincident field at co generates a fixed-amplitude LO at 2ω in the KTPfilm which is truly “local” to the surface. This field then combineswith the SH providing a >100× enhancement in the SH response. Theadsorption of bovine serum albumin (BSA) to the sensor surface was usedto measure the achievable gain of these novel KTP-LO films. The goal ofAim #1 is to design and optimize the heterodyne system by varying thenonlinear optical layer thickness and tuning for optimal amplificationby varying the thickness of the SiO₂ over-layer. The phase difference(ϕ) between the LO and SH field is given by (ϕ)=2πnd/λ_(o), where n isthe refractive index of the SiO₂ layer, d is the thickness and λ_(o) isthe SH wavelength in a vacuum. It is contemplated that a spectroscopyellipsometer can aid in the fabrication and characterization of thesensor platform. Based on our preliminary findings, the amplificationstrategy disclosed herein effectively decreases the LOD of SH by 1-2orders of magnitude (LOD=3π/sensitivity), bringing us into the sub-fMand atoM detection regime. This amplification strategy can be applied toSHCS and SHI, thus enhancing the capabilities of these methods for thedetection of cancer biomarkers, as described below.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

REFERENCES

1. Smith K A, Gale B K, Conboy J C. Micropatterned Fluid Lipid BilayerArrays Created Using a Continuous Flow Microspotter. AnalyticalChemistry 2008; 80:7980-7.2. Conboy J C, Kriech M A. Measuring melittin binding to planarsupported lipid bilayer by chiral second harmonic generation. Anal ChimActa. 2003; 496:143-53.3. Evans-Nguyen K M, Fuierer R R, Fitchett B D, Tolles L R, Conboy J C,Schoenfisch M H. Changes in Adsorbed Fibrinogen upon Conversion toFibrin. Langmuir. 2006; 22:5115-21.4. Kriech M A, Conboy J C. Using the intrinsic chirality of a moleculeas a label-free probe to detect molecular adsorption to a surface bysecond harmonic generation. Appl Spectrosc. 2005; 59:746-53.5. Nguyen T T, Sly K L, Conboy J C. Comparison of the Energetics ofAvidin, Streptavidin, NeutrAvidin, and Anti-Biotin Antibody Binding toBiotinylated Lipid Bilayer Examined by Second-Harmonic Generation.Analytical Chemistry 2012; 84:201-8.6. Nguyen T T, Rembert K, Conboy J C. Label-Free Detection ofDrug-Membrane Association Using Ultraviolet-Visible Sum-FrequencyGeneration. Journal of the American Chemical Society. 2009; 131:1401-3.7. Stokes G, Conboy John C. Measuring Selective Estrogen ReceptorModulator (SERM)-Membrane Interactions with Second Harmonic Generation.Journal of the American Chemical Society. 2014; 136:1409-1417.8. Nguyen T T, Conboy J C. High-Throughput Screening of Drug-LipidMembrane Interactions via Counter-Propagating Second Harmonic GenerationImaging. Analytical Chemistry 2011; 83:5979-88.9. Sly K L, Nguyen T T, Conboy J C. Lens-less surface second harmonicimaging. Opt Express. 2012; 20:21953-67.10. Sly K L, Mok S-W, Conboy J C. Second Harmonic CorrelationSpectroscopy: A Method for Determining Surface Binding Kinetics andThermodynamics. Analytical Chemistry. 2013; 85:8429-35.

11. Sly K L, Conboy John C. Determination of Multivalent Protein-LigandBinding Kinetics and Energetics Using Second Harmonic CorrelationSpectroscopy Analytical Chemistry 2014; 86:11045-11054.

12. Kazane S A, Sok D, Cho E H, Uson M L, Kuhn P, Schultz P G, et al.Site-specific DNA-antibody conjugates for specific and sensitiveimmuno-PCR. Proceedings of the National Academy of Sciences of theUnited States of America. 2012; 109:3731-6.13. Garcia-Schwarz G, Santiago J G. Rapid High-Specificity microRNADetection Using a Two-stage Isotachophoresis Assay. Angew Chem, Int Ed.2013; 52:11534-7.14. Law W-C, Yong K-T, Baev A, Prasad P N. Sensitivity Improved SurfacePlasmon Resonance Biosensor for Cancer Biomarker Detection Based onPlasmonic Enhancement. ACS Nano. 2011; 5:4858-64.15. Krishnan S, Mani V, Wasalathanthri D, Kumar C V, Rusling J F.Attomolar Detection of a Cancer Biomarker Protein in Serum by SurfacePlasmon Resonance Using Superparamagnetic Particle Labels. Angew Chem,Int Ed. 2011; 50:1175-8, S/1-S/4.16. Samanta A, Maiti K K, Soh K-S, Liao X, Vendrell M, Dinish U S, etal. Ultrasensitive Near-Infrared Raman Reporters for SERS-Based In VivoCancer Detection. Angew Chem, Int Ed. 2011; 50:6089-92, S/1-S/23.17. Panikkanvalappil S R, Mackey M A, El-Sayed M A. Probing the UniqueDehydration-Induced Structural Modifications in Cancer Cell DNA UsingSurface Enhanced Raman Spectroscopy. Journal of the American ChemicalSociety. 2013; 135:4815-21.18. Li M, Cushing S K, Zhang J, Suri S, Evans R, Petros W P, et al.Three-Dimensional Hierarchical Plasmonic Nano-Architecture EnhancedSurface-Enhanced Raman Scattering Immunosensor for Cancer BiomarkerDetection in Blood Plasma. ACS Nano. 2013; 7:4967-76.19. Wu G, Datar R H, Hansn K M, Thundat T, Cote R J, Majumdar A.Bioassay of prostate-specific antigen (PSA) using microcantilevers. NatBiotechnol. 2001; 19:856-60.20. Loo L N, Capobianco J A, Wu W, Gao X, Shih W Y, Shih W-H, et al.Highly sensitive detection of HER2 extracellular domain in the serum ofbreast cancer patients by piezoelectric microcantilevers. AnalyticalChemistry (Washington, DC, United States). 2011; 83:3392-7.21. Wang J, Wu L, Ren J, Qu X. Visualizing Human Telomerase Activitywith Primer-Modified Au Nanoparticles. Small. 2012; 8:259-64.22. Song Y, Wei W, Qu X. Colorimetric Biosensing Using Smart Materials.Adv Mater (Weinheim, Ger). 2011; 23:4215-36.23. Labib M, Khan N, Ghobadloo S M, Cheng J, Pezacki J P, Berezovski MV. Three-Mode Electrochemical Sensing of Ultralow MicroRNA Levels.Journal of the American Chemical Society. 2013; 135:3027-38.24. Chikkaveeraiah B V, Bhirde A A, Morgan N Y, Eden H S, Chen X.Electrochemical Immunosensors for Detection of Cancer ProteinBiomarkers. ACS Nano. 2012; 6:6546-61.25. Rana S, Singla A K, Bajaj A, Elci S G, Miranda O R, Mout R, et al.Array-Based Sensing of Metastatic Cells and Tissues UsingNanoparticle-Fluorescent Protein Conjugates. ACS Nano. 2012; 6:8233-40.26. Mizusawa K, Takaoka Y, Hamachi I. Specific Cell Surface ProteinImaging by Extended Self-Assembling Fluorescent Turn-on Nanoprobes.Journal of the American Chemical Society. 2012; 134:13386-95.27. Wu L, Qu X. Cancer biomarker detection: recent achievements andchallenges. Chem Soc Rev. 2015; 44:2963-97.28. Rusling J F, Kumar C V, Gutkind J S, Patel V. Measurement ofbiomarker proteins for point-of-care early detection and monitoring ofcancer. Analyst (Cambridge, UK). 2010; 135:2496-511.29. Li J, Li S, Yang C F. Electrochemical biosensors for cancerbiomarker detection. Electroanalysis. 2012; 24:2213-29.30. Luo X, Davis J J. Electrical biosensors and the label free detectionof protein disease biomarkers. Chem Soc Rev. 2013; 42:5944-62.31. Swierczewska M, Liu G, Lee S, Chen X. High-sensitivity nanosensorsfor biomarker detection. Chem Soc Rev. 2012; 41:2641-55.32. de la Rica R, Stevens M M. Plasmonic ELISA for the ultrasensitivedetection of disease biomarkers with the naked eye. Nat Nanotechnol.2012; 7:821-4.33. Alberti D, Erve Mvt, Stefania R, Ruggiero M R, Tapparo M, GeninattiCrich S, et al. A Quantitative Relaxometric Version of the ELISA Testfor the Measurement of Cell Surface Biomarkers. Angew Chem, Int Ed.2014; 53:3488-91.34. Reen D J. Enzyme-linked immunosorbent assay (ELISA). Methods MolBiol (Totowa, N.J.). 1994; 32:461-6.35. Jackson T M, Ekins R P. Theoretical limitations on immunoassaysensitivity: Current practice and potential advantages of fluorescentEu3+ chelates as non-radioisotopic tracers. Journal of ImmunologicalMethods. 1986; 87(1):13-20.

36. Shen Y R. The Principles of Nonlinear Optics: John Wiley and Sons,Inc; 1984.

37. Stokes G, Conboy John C. Measuring Selective Estrogen ReceptorModulator (SERM)-Membrane Interactions with Second Harmonic Generation.JACS. 2014; 136(4):1409-17.38. Kriech M A, Conboy J C. Label-free chiral detection of melittinbinding to a membrane. Journal of the American Chemical Society. 2003;125:1148-9.39. Dai H-L, Zeng J, editors. Real-time resolved observation ofmolecular transport through living cell membranes by optical secondharmonic generation 2008: American Chemical Society.40. Muller U R, Nicolau D V, editors. Microarray Technology and ItsApplications. Berlin Springer-Verlag; 2005.41. Falciani F, editor. Microarray Technology Through Applications. NewYork: Taylor and Francis Group; 2007.42. Zhang F, Gates R J, Smentkowski V S, Natarajan S, Gale B K, Watt RK, et al. Direct Adsorption and Detection of Proteins, IncludingFerritin, onto Microlens Array Patterned Bioarrays. Journal of theAmerican Chemical Society. 2007; 129(30):9252-3.43. Chang-Yen D A, Myszka D, Gale B K A. A novel PDMS microfluidicspotter for fabrication of protein chips and microarrays. Proc SPIE.2005; 5718:111.44. Natarajan S, Hatch A, Myszka D G, Gale B K. Optimal Conditions forProtein Array Deposition Using Continuous Flow. Anal Chem. 2008;80:8561-7.45. Eddings MAea. Improved continuous-flow print head for micro-arraydeposition. Anal Biochem. 2008; 382:55-9.46. Grunwell J R, Glass J L, Lacoste T D, Deniz A A, Chemla D S, SchultzP G. Monitoring the Conformational Fluctuations of DNA Hairpins UsingSingle-Pair Fluorescence Resonance Energy Transfer. Journal of theAmerican Chemical Society. 2001; 123(18):4295-303.47. Wennmalm S, Edman L, Rigler R. Conformational fluctuations in singleDNA molecules. Proceedings of the National Academy of Sciences of theUnited States of America. 1997; 94(20):10641-6.48. Ladd J, Boozer C, Yu Q, Chen S, Homola J, Jiang S. DNA-DirectedProtein Immobilization on Mixed Self-Assembled Monolayers via aStreptavidin Bridge. Langmuir. 2004; 20(19):8090-5.49. Esseghaier C, Helali S, Ben Fredj H, Tlili A, Abdelghani A.Polypyrrole-neutravidin layer for impedimetric biosensor. Sensors andActuators, B: Chemical. 2008; B131(2):584-9.50. Sun H, Choy T S, Zhu D R, Yam W C, Fung Y S. Nano-silver-modifiedPQC/DNA biosensor for detecting E. coli in environmental water.Biosensors & Bioelectronics. 2009; 24(5):1405-10.51. Hall W P, Ngatia S N, Van Duyne R P. LSPR Biosensor SignalEnhancement Using Nanoparticle-Antibody Conjugates. Journal of PhysicalChemistry C. 2011; 115(5):1410-4.52. Bashir R, Gomez R, Sarikaya A, Ladisch M R, Sturgis J, Robinson J P.Adsorption of avidin on microfabricated surfaces for protein biochipapplications. Biotechnology and Bioengineering. 2001; 73(4):324-8.53. Lazcka O, Del Campo F J, Munoz F X. Pathogen detection: Aperspective of traditional methods and biosensors. Biosensors &Bioelectronics. 2007; 22(7):1205-17.54. Barton A C, Davis F, Higson S P J. Labeless Immunosensor Assay forthe Stroke Marker Protein Neuron Specific Enolase Based upon anAlternating Current Impedance Protocol. Analytical Chemistry(Washington, D.C., United States). 2008; 80(24):9411-6.55. Zhavnerko G K, Yi S J, Chung S H, Yuk J S, Ha K S. Orientedimmobilization of C-reactive protein on solid surface for biosensorapplications. NATO Science Series, II: Mathematics, Physics andChemistry. 2004; 152(Frontiers of Multifunctional IntegratedNanosystems):95-108.56. Cooper M A. Optical biosensors in drug discovery. Nature ReviewsDrug Discovery. 2002; 1(7):515-28.57. Teeter J S, Meyerhoff R D. Environmental fate and chemistry ofraloxifene hydrochloride. Environmental Toxicology and Chemistry. 2002;21(4):729-36.58. Barrett-Connor E, Mosca L, Collins P, Geiger M J, Grady D, KornitzerM, et al. Effects of Raloxifene on Cardiovascular Events and BreastCancer in Postmenopausal Women. New England Journal of Medicine. 2006;355(2):125-37.59. Dodge J A, Lugar C W, Cho S, Short L L, Sato M, Yang N N, et al.Evaluation of the major metabolites of raloxifene as modulators oftissue selectivity. The Journal of Steroid Biochemistry and MolecularBiology. 1997; 61(1-2):97-106.60. Dutertre M, Smith C L. Molecular Mechanisms of Selective EstrogenReceptor Modulator (SERM) Action. Journal of Pharmacology andExperimental Therapeutics. 2000; 295(2):431-7.61. Morello K C, Wurz G T, DeGregorio M W. Pharmacokinetics of SelectiveEstrogen Receptor Modulators. Clinical Pharmacokinetics. 2003;42(4):361-72.62. Magde D, Elson E, Webb W W. Thermodynamic fluctations in a reactingsystem. Measurement by fluorescence correlation spectroscopy. PhysicalReview Letters. 1972; 29(11):705-8.63. Koppel D E, Axelrod D, Schlessinger J, Elson E L, Webb W W. Dynamicsof fluorescence marker concentration as a probe of mobility. BiophysicalJournal. 1976; 16(11):1315-29.64. Magde D, Elson E L, Webb W W. Fluorescence correlation spectroscopy.II. Experimental realization. Biopolymers. 1974; 13(1):29-61.65. Thompson N L, Navaratnarajah P, Wang X. Measuring Surface BindingThermodynamics and Kinetics by Using Total Internal Reflection withFluorescence Correlation Spectroscopy: Practical Considerations. Journalof Physical Chemistry B. 2011; 115(1):120-31.66. Starr T E, Thompson N L. Total internal reflection with fluorescencecorrelation spectroscopy: combined surface reaction and solutiondiffusion. Biophysical Journal. 2001; 80(3):1575-84.67. Hansen R L, Harris J M. Measuring reversible adsorption kinetics ofsmall molecules at solid/liquid interfaces by total internal reflectionfluorescence correlation spectroscopy. Anal Chem. 1998; 70(20):4247-56.68. Thompson N L, Burghardt T P, Axelrod D. Measuring surface dynamicsof biomolecules by total internal reflection fluorescence withphotobleaching recovery or correlation spectroscopy. BiophysicalJournal. 1981; 33(3):435-54.69. Maiti S, Haupts U, Webb W W. Fluorescence correlation spectroscopy:diagnostics for sparse molecules. Proceedings of the National Academy ofSciences of the United States of America. 1997; 94(22):11753-7.70. Watanabe H, Yamaguchi S, Sen S, Morita A, Tahara T. “Half-hydration”at the air/water interface revealed by heterodyne-detected electronicsum frequency generation spectroscopy, polarization second harmonicgeneration, and molecular dynamics simulation. J Chem Phys. 2010;132:144701/1-/9.71. Golovan L A, Melnikov V A, Bestem'Yanov K P, Zabotnov S V, GordienkoV M, Timoshenko V Y, et al. Disorder-correlated enhancement ofsecond-harmonic generation in strongly photonic porous galliumphosphide. Applied Physics B: Lasers and Optics. 2005; 81(2-3):353-6.72. Frostell-Karlsson A, Remaeus A, Roos H, Andersson K, Borg P,Haemaelaeinen M, et al. Biosensor Analysis of the Interaction betweenImmobilized Human Serum Albumin and Drug Compounds for Prediction ofHuman Serum Albumin Binding Levels. Journal of Medicinal Chemistry.2000; 43(10):1986-92.73. Murtaza R, Jackman H L, Alexander B, Lleshi-Tali A, Winnie A P, IgicR. Simultaneous determination of mepivacaine, tetracaine, andp-butylaminobenzoic acid by high-performance liquid chromatography.Journal of Pharmacological and Toxicological Methods. 2001; 46(3):131-6.74. Haes A J, Van Duyne R P. A nanoscale optical biosensor: Sensitivityand selectivity of an approach based on the localized surface plasmonresonance spectroscopy of triangular silver nanoparticles. Journal ofthe American Chemical Society. 2002; 124(35):10596-604.75. Zhao S, Walker D S, Reichert W M. Cooperativity in the binding ofavidin to biotin-lipid-doped Langmuir-Blodgett films. Langmuir. 1993;9(11):3166-73.76. Shi J, Yang T, Kataoka S, Zhang Y, Diaz A J, Cremer PS. GM1Clustering Inhibits Cholera Toxin Binding in Supported PhospholipidMembranes. Journal of the American Chemical Society. 2007;129(18):5954-61.77. Pierce Biotechnologies I. [cited]; Available from:http://www.piercenet.com/guide/guide-elisa-substrates.78. Vornholt W, Hartmann M, Keusgen M. SPR studies ofcarbohydrate-lectin interactions as useful tool for screening on lectinsources. Biosensors and Bioelectronics. 2007; 22(12):2983-8.79. Jung H, Yang T, Lasagna M D, Shi J, Reinhart G D, Cremer P S. Impactof hapten presentation on antibody binding at lipid membrane interfaces.Biophysical Journal. 2008; 94(8):3094-103.

80. Sino Biological I.

81. Hirano S-I, Yogo T, Kikuta K-I, Noda K-I, Ichida M, Nakamura A.Synthesis of KTiOPO4 (KTP) thin films using metallo-organics. Journal ofthe American Ceramic Society. 1995; 78(11):2956-60.82. Li D, Kong L, Zhang L, Yao X. Sol-gel preparation andcharacterization of transparent KTiOPO4/SiO2 nanocomposite glass forsecond harmonic generation. Journal of Non-Crystalline Solids. 2000;271(1,2):45-55.83. Polanski M, Anderson N L. A List of Candidate Cancer Biomarkers forTargeted Proteomics. Biomarker Insights. 2006; 1:1-48.84. Cook G B, Neaman I E, Goldblatt J L, Cambetas D R, Hussain M,Luftner D, et al. Clinical utility of serum HER-2/neu testing on thebayer Immuno 1 automated system in breast cancer. Anticancer Research.2001; 21(2B):1465-70.85. Gann P H, Hennekens C H, Stampfer M J. A prospective evaluation ofplasma prostate-specific antigen for detection of prostatic cancer.JAMA: the journal of the American Medical Association. 1995;273(4):289-94.86. Yamaguchi K, Nagano M, Torada N, Hamasaki N, Kawakita M, Tanaka M.Urine diacetylspermine as a novel tumor marker for pancreatobiliarycarcinomas. Rinsho Byori. 2004; 52:336-9.87. Ciambellotti E, Coda C, Lanza E. Determination++ of CA 15-3 in thecontrol of primary and metastatic breast carcinoma. Minerva Med. 1993;84 (Copyright (C) 2013 U.S. National Library of Medicine.):107-12.88. Mor G, Visintin I, Lai Y, Zhao H, Schwartz P, Rutherford T, et al.Serum protein markers for early detection of ovarian cancer. Proceedingsof the National Academy of Sciences of the United States of America.2005; 102:7677-82.89. Xiao T, Ying W, Li L, Hu Z, Ma Y, Jiao L, et al. An approach tostudying lung cancer-related proteins in human blood. Mol CellProteomics. 2005; 4:1480-6.90. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. CancerStatistics, 2008. CA: A Cancer Journal for Clinicians. 2008;58(2):71-96.91. Rhea J M, Molinaro R J. Cancer biomarkers: surviving the journeyfrom bench to bedside. MLO: medical laboratory observer. 2011;43(3):10-2, 6, 8; quiz 20, 2.92. Polanski M, Anderson N. A list of candidate cancer biomarkers fortargeted proteomics. Biomarker Insights. 2007; 1:1-48.93. Mattson G, Conklin E, Desai S, Nielander G, Savage M D, Morgensen S.A practical approach to crosslinking. Molecular Biology Reports. 1993;17(3):167-83.94. Yoshitake S, Imagawa M, Ishikawa E, Niitsu Y, Urushizaki I, NishiuraM, et al. Mild and efficient conjugation of rabbit Fab′ and horseradishperoxidase using a maleimide compound and its use for enzymeimmunoassay. Journal of Biochemistry. 1982; 92(5):1413-24.95. Liu J, Eddings M A, Miles A R, Bukasov R, Gale B K, Shumaker-Parry JS. In situ microarray fabrication and analysis using a microfluidic flowcell array integrated with surface plasmon resonance microscopy. AnalChem. 2009; 81:4296-301.96. Natarajan S, Hatch A, Myszka David G, Gale Bruce K. Optimalconditions for protein array deposition using continuous flow. AnalChem. 2008; 80(22):8561-7.

We claim:
 1. A method of screening for analytes that bind an antibody orantibody fragment comprising a) introducing an analyte to an antibody orantibody fragment; and b) detecting the presence of the analyte bound tothe antibody or antibody fragment using a label-free second harmonicdetection system, wherein the presence of the analyte bound to theantibody or antibody fragment indicates the analyte binds the antibodyor antibody fragment.
 2. The method of claim 1, wherein the antibody orantibody fragment is immobilized on a support.
 3. The method of claim 1,wherein the antibody or antibody fragment is known to bind the analyteof interest.
 4. The method of claim 1, wherein the analyte is a protein,nucleic acid, small molecule, or fragment thereof.
 5. The method ofclaim 1, wherein detecting the presence of the analyte bound to theantibody or antibody fragment using the label-free second harmonicdetection system comprises using second harmonic imaging (SHI) to detectthe binding of the analyte to the antibody or antibody fragment.
 6. Themethod of claim 5, wherein detecting the presence of the analyte boundto the antibody or antibody fragment using the label-free secondharmonic detection system further comprises determining bindingproperties of the analyte of interest using second-harmonic correlationspectroscopy.